13C NMR spectroscopy is a powerful tool that can potentially provide quantitative, clinically relevant data about a variety of metabolic pathways. The 13C isotopomer methods we have developed over the past 6 years are reaching fruition and we are now in a position to begin applying these methods in live animals, as a prelude to eventual human clinical studies. One technical issue which may ultimately limit the application of 13C isotopomer methods in vivo is resolution of the multi-lined resonances which contain the isotopomer information. We have recently shown that (13C)homonuclear decoupling of a single glutamate resonance collapses neighboring nine-line resonances into 3-line multiplets, which can be resolved in intact tissue. Two other advantages result, signal-to noise is improved and 13C isotopomer information is preserved. As a result, we are now able to monitor relative oxidation of a mixture of three substrates in intact tissue by collecting a single 13C spectrum. We propose to use this method to monitor oxidation among fatty acids, lactate and ketone bodies in two different. heart models, the perfused rat heart and in vivo rabbit heart (closed chest model). We will also monitor competitive substrate utilization, measure gluconeogenic flux relative to citric acid cycle flux using isotopomer methods, and test the hypothesis that glucose and glutamate are derived from a common pool of oxaloacetate in perfused mouse liver. We will attempt to use 13C magnetization transfer techniques to quantitate citric acid cycle flux or flux through pathways related to the cycle, such as the malate-aspartate transport shuttle or the transaminases. Finally, we will use localized 13C NMR spectroscopy (with (13C)homonuclear decoupling) to obtain an index of citric acid cycle flux in the in vivo rabbit myocardium and monitor utilization of fatty acids, lactate and ketone bodies in hearts of closed- chest animals during changes in oxygen consumption. Our long-term goal is to demonstrate that 13C multiplets can be resolved in the in vivo myocardium, that clinically useful metabolic information may be derived from single spectra, and that 13C isotopomer analyses may eventually be possible in humans.